Involvement of DPP‐IV catalytic residues in enzyme–saxagliptin complex formation
2008
The inhibition of DPP-IV by
saxagliptinhas been proposed to occur through formation of a covalent but reversible complex. To evaluate further the mechanism of inhibition, we determined the X-ray crystal structure of the DPP-IV:saxagliptin complex. This structure reveals covalent attachment between S630 and the inhibitor
nitrilecarbon (C–O distance <1.3 A). To investigate whether this serine addition is assisted by the catalytic His-Asp dyad, we generated two mutants of DPP-IV, S630A and H740Q, and assayed them for ability to bind inhibitor. DPP-IVH740Q bound
saxagliptinwith an ∼1000-fold reduction in affinity relative to DPP-IVWT, while DPP-IVS630A showed no evidence for binding inhibitor. An analog of
saxagliptinlacking the
nitrilegroup showed unchanged binding properties to the both
mutant proteins, highlighting the essential role S630 and H740 play in covalent bond formation between S630 and
saxagliptin. Further supporting mechanism-based inhibition by
saxagliptin, NMR spectra of enzyme–
saxagliptincomplexes revealed the presence of three downfield resonances with low fractionation factors characteristic of short and strong hydrogen bonds (SSHB). Comparison of the NMR spectra of various wild-type and mutant DPP-IV:ligand complexes enabled assignment of a resonance at ∼14 ppm to H740. Two additional DPP-IV mutants, Y547F and Y547Q, generated to probe potential stabilization of the enzyme–inhibitor complex by this residue, did not show any differences in inhibitor binding either by ITC or NMR. Together with the previously published enzymatic data, the structural and binding data presented here strongly support a histidine-assisted covalent bond formation between S630 hydroxyl oxygen and the
nitrilegroup of
saxagliptin.
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